Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease characterized by degeneration of spinal cord anterior horn cells, which lead to muscular paralysis with muscular atrophy. SMA patients are afflicted to varying degrees of severity and therefore clinically categorized as type 1 (severe), 2 (intermediate), or 3 (mild), according to age of onset and rate of progression. The disorder is found in approximately 1 in 10,000 live births and has a carrier frequency of 1 in 50 (Zerres (1997) J. Neurol. Sci. 146:67-72). Type 1 patients have a life expectancy of 18 months or less, whereas type 3 patients can survive into adulthood.
All types of human spinal muscular atrophy are due to mutations in the SMN1 gene of the 5q13 locus on chromosome 5. In most individuals, there exists a second gene, SMN2, adjacent to SMN1. Both SMN1 and SMN2 encode SMN, a 294 amino acid RNA-binding protein (Lefebvre et al. (1995) Cell 80:155-165; Monani et al. (1999) Hum. Mol. Genet. 8:1177-1183). At the genomic level, only five nucleotides have been found that differentiate the SMN1 gene from the SMN2 gene. Furthermore, the two genes produce identical mRNAs, except for a silent nucleotide change in exon 7, namely, a Cxe2x86x92T change six base pairs inside exon 7 in SMN2 as compared to SMN1. This mutation modulates the activity of an exon splicing enhancer (Lorson and Androphy (2000) Hum. Mol. Genet. 9:259-265). The result of this and the other nucleotide changes in the intronic and promoter regions is that most SMN2 transcripts lack exons 3, 5, or 7. In contrast, the mRNA transcribed from the SMN1 gene is generally a full-length mRNA with only a small fraction of its transcripts spliced to remove exon 3, 5, or 7 (Gennarelli et al. (1995) Biochem. Biophys. Res. Commun. 213:342-348; Jong et al. (2000) J. Neurol. Sci. 173:147-153).
Furthermore, there is substantially less transcription of SMN2 than SMN1 in most individuals. As the severity of deletions of the SMN1 indicates, the low level of full-length SMN protein produced by SMN2 is insufficient to protect against spinal muscular atrophy disease (Lefebvre, supra; Coovert et al. (1997) Hum. Mol. Genet. 6:1205-1214).
There is no effective treatment to date for spinal muscular atrophy disease.
The invention is based on the discovery that different classes of compounds have been identified, using new methods, as being useful in the modulation of SMN exon 7 gene expression, and therefore as being useful in the treatment of SMA. It has also been discovered that cells harvested from SMA patients and transgenic animals having particular genotypes and phenotypes are useful in the new screening methods.
Accordingly, the invention features a method for modulating SMN gene expression in a subject. The method includes administering to the subject an amount of hydroxyurea sufficient to increase the expression level of SMN exon 7 in a cell of the subject, relative to a reference expression level of SMN exon 7. For example, the method can increase the ratio of SMN transcripts having exon 7 to those lacking exon 7 by at least 30, 50, 60, 70, 80, 90, 100, or 150%. The cell can be a mammalian cell, e.g., a human cell, e.g., a cell from a human SMA patient. For example, the cell can be homozygous for an SMN1 mutation.
In another aspect, the invention features a method of treating spinal muscular atrophy in a subject. The method includes administering to the subject hydroxyurea in an amount sufficient to ameliorate a symptom of spinal muscular atrophy. The subject can be human, e.g., a human homozygous for mutations in SMN1. The ameliorated symptom can be muscular paralysis, muscular atrophy, breathing, walking gait, or a symptom described herein. The symptom can also be decreased expression of SMN exon 7 in a cell of the subject. The administered amount can be about 0.2 to 100, 0.4 to 80, 5 to 50, or 15 to 25 mg/kg/day. The amount can be administered orally or parentally.
In one embodiment, the subject is a fetus, and the hydroxyurea is administered to the subject in utero.
In still another aspect, the invention features a method of for modulating SMN gene expression in a cell. The method includes contacting an amount of hydroxyurea to the cell. The amount is sufficient to increase the expression level of SMN exon 7 in the cell, relative to a reference expression level of SMN exon 7. The cell can be a human cell, e.g., a cell in a patient. The cell can treated in vivo, in vitro, or ex vivo. Following ex vivo treatment, the cell can be administered (e.g., restored) to a subject, e.g., a human patient.
In yet another aspect, the invention features a method of treating spinal muscular atrophy in a subject. The method includes administering to the subject a DNA replication inhibitor, e.g., a ribonucleotide reductases inhibitor (e.g., hydroxyurea or a compound other than hydroxyurea) in an amount sufficient to ameliorate a symptom of spinal muscular atrophy. The subject can be human, e.g., a human homozygous for mutations in SMN1. The ameliorated symptom can be muscular paralysis, muscular atrophy, breathing, walking gait, or a symptom described herein. The symptom can also be decreased expression of SMN exon 7 in a cell of the subject.
As used herein, the term xe2x80x9ctransgenexe2x80x9d refers to a nucleic acid sequence (e.g., encoding one or more human proteins), which is inserted by artifice into a cell. The transgene is integrated into a chromosomal genome. A transgenic sequence can be partly or entirely species-heterologous, i.e., the transgenic sequence, or a portion thereof, can be from a species which is different from the cell into which it is introduced. A transgenic sequence can be partly or entirely species-homologous, i.e., the transgenic sequence, or a portion thereof, can be from the same species as is the cell into which it is introduced. If a transgenic sequence is homologous (in the sequence sense or in the species-homologous sense) to an endogenous gene of the cell into which it is introduced, then the transgenic sequence has one or more of the following characteristics: it is designed for insertion, or is inserted, into the cell""s genome in such a way as to alter the sequence of the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the endogenous gene or its insertion results in a change in the sequence of the endogenous gene); it includes a mutation, e.g., a mutation which results in misexpression of the transgenic sequence; by virtue of its insertion, it can result in misexpression of the gene into which it is inserted, e.g., the insertion can result in a knockout of the gene into which it is inserted. A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid sequences, such as introns, that may be necessary for a desired level or pattern of expression of a selected nucleic acid. A transgene can provide an antisense transcript or a sense transcript, e.g., a transcript encoding a protein.
As used herein, the term xe2x80x9ctransgenic cellxe2x80x9d refers to a cell containing a transgene.
As used herein, a xe2x80x9ctransgenic animalxe2x80x9d is a non-human animal in which one or more (e.g., all) of the cells of the animal contain a heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques known in the art. The transgene can be introduced into the cell directly, indirectly by introduction into a precursor of the cell, or by way of deliberate genetic manipulation, such as by microinjection, transformation, electroporation, lipofection, or infection with a recombinant virus. In one example, where the transgene is introduced indirectly, the transgene is introduced into a cultured cell, and the nucleus of the cultured cell or of a descendant of the cultured cell is microinjected into an enucleated oocyte to produce a nucleated oocyte which develops into an animal.
As used herein, a xe2x80x9cdisruptionxe2x80x9d in reference to an endogenous gene refers to any type of mutation that inactivates an endogenous gene, an exon thereof, or the amino acid sequence encoded by the endogenous gene or exon thereof. Consequently, the mutation can be a deletion of the disrupted gene or portion thereof, a mutation that causes inappropriate splicing (including abolishment of splicing), and/or and insertion into the disrupted gene or portion thereof.
In reference to animal models (e.g., a transgenic mouse model) of SMA, a symptom of SMA is selected from: lethality before birth, before postnatal day 10, or before 4 weeks of age; decreased fetal movement; lethargy; loss or depression of muscular reflexes (e.g., areflexia, loss of gag reflex); hand tremors; peripheral neuropathies; large amplitude, prolonged, polyphasic discharges on active muscle contraction as detected by EMG (electromyography); myopathies; muscular weakness (e.g., weakness in the pelvic girdle, arms, facial muscles, instability of walking gait, paralysis of hind limbs, tongue fasciculation, and atrophy); myasthenia; hypertrophied muscle bundles (e.g., pseudohypertrophy of the calves); fat infiltration in muscle bundles; fibrosis in muscle bundles; necrosis in muscle bundles; muscular dystrophies; atrophy of muscle bundles (e.g., in tail, trunk, or limbs); decreased diameter of muscle fibers in the tail, trunk, or limbs; shorter and enlarged tails; chronic necrosis of the tail tip; subcutaneous edema; and reduced furry coat hair;
The skilled artisan can readily determine which of the list of symptoms would apply to a particular animal model. For example, a shortened tail is relevant only to those animals having a tail, and hand tremors are only relevant to those animals having a hand (e.g., a primate.). A symptom for type 1 muscular atrophy in a mouse includes lethality before postnatal day 10, reduced furry coat hair, and a shortened and enlarged tail.
A used herein, the term xe2x80x9cmodulatingxe2x80x9d refers to a change in level, either an increase or a decrease. The change can be detected in a qualitative or quantitative observation. If a quantitative observation is made, and if a comprehensive analysis is performed over a plurality of observations, one skilled in the art can apply routine statistical analysis to identify modulations where a level is changed and where the statistical parameter, the p value is less than 0.05.
As used herein, xe2x80x9cfull length SMN gene expressionxe2x80x9d or xe2x80x9cexpression level of SMN exon 7xe2x80x9d refers to a scenario where an SMN gene is transcribed and the resulting transcripts contain exon 7 of an SMN gene. Specifically, it is of no consequence whether the exon 7-containing transcript is transcribed from the human SMN1 gene or from the human SMN2 gene. Transcripts containing SMN exon 7 are translated into the 294 amino acid SMN polypeptide. The amino acid sequence of the 294 amino acid SMN polypeptide is described in GenBank entry xe2x80x9cGI:624186.xe2x80x9d The nucleic acid sequence of SMN exon 7 is the sequence contained between nucleotides about 868 and about 921 of GenBank entry xe2x80x9cGI:624185.xe2x80x9d The identify of the sixth base of exon 7 can be C (cytosine) if the transcript is derived from SMN1 or U (uracil) if the transcript is derived from SMN2. Exon 7 expression can be analyzed in cells in which SMN1 is deleted or mutated. Thus, the relevant SMN exon 7 sequence contains a uracil at position 873 while the remainder of the sequence is as recited from nucleotides about 868 to about 921 of GenBank entry xe2x80x9cGI:624185.xe2x80x9d
As used herein, a xe2x80x9cribonucleotide reductases inhibitorxe2x80x9d is a compound that has an IC50 of less than 800 xcexcM for inhibition of a mammalian ribonucleotide reductases in an in vitro assay. One exemplary in vitro assay is described in U.S. Pat. No. 5,071,835. Further assays are described in Elford et al. (1981) Adv. Enz. Reg., 19, 151 and Cohen (1985) J. Gen. Virol. 66:733.
A xe2x80x9cDNA replication inhibitorxe2x80x9d is a compound that when contacted to mammalian cells causes at least 80% of the cells to arrest prior to or in S phase after 36 hours. The position of cells in the cell cycle can be determine by fluorescent activated cell sorting.
Other features, objects, and advantages of the invention will be apparent from the description and from the claims.